SUMOs are Ubiquitin-like proteins, with diverse roles in nuclear function. Vertebrate cells express three major SUMO paralogues (SUMO-1-3): Mature SUMO-2 and -3 are 95% identical to each other, while SUMO-1 is 45% identical to SUMO-2 or -3. (Where they are functionally indistinguishable, SUMO-2 and &#8722;3 will be collectively called SUMO-2/3.) SUMOylation is dynamic due to rapid turnover of conjugated species by SUMO proteases. Both processing and deSUMOylation are mediated by the same family of proteases (called Ubl specific proteases (Ulp) in yeast and Sentrin-specific proteases (SENP) in vertebrates), which play a pivotal role in determining the spectrum of SUMOylated species. There are two yeast Ulps (Ulp1p and Ulp2p/Smt4p), and six mammalian SENPs (SENP1, 2, 3, 5, 6, and 7). SENP1, 2, 3 and 5 form a Ulp1p-related sub-family, while SENP6 and 7 are more closely related to Ulp2p. Yeast Ulps have important roles in mitotic progression and chromosome segregation;we wished to determine which SENPs played similar roles in metazoans. Toward this end, we have focused on characterization of vertebrate SENPs. We have defined the enzymatic specificity of these proteins and analyzed their key biological roles, particularly the functions of SENP3 and SENP5 in ribosome biogenesis and the function of SENP6 in kinetochore assembly. Ulp1p localizes to nuclear pores and is encoded by an essential gene;it is important for SUMO processing, nucleocytoplasmic trafficking and late steps in the ribosome biogenesis pathway. Ribosome biogenesis occurs largely within the nucleolus. It is a major metabolic expense and a critical point of cellular regulation during both cell growth and cancer progression. This process has been studied genetically in yeast, but remains poorly characterized in metazoans. Human SENP3 and SENP5 are nucleolar Ulp1p-like SUMO proteases that are closely related to each other, and that have enzymatic specificity for SUMO-2/3 over SUMO-1. We examined the subnucleolar localization, behavior, and function of SENP3 and SENP5. Both of these enzymes colocalized and physically interacted with B23/nucleophosmin, an abundant 37-kD phosphoprotein that shuttles between the nucleolus and cytoplasm. B23/nucleophosmin is implicated in many cellular processes, including ribosome biogenesis and control of the Arf-MDM2-p53 pathway;it is often overexpressed in solid tumors and has been strongly linked to hematopoietic malignancies. We found that B23/nucleophosmin is essential for the stable accumulation of SENP3 and SENP5. After depletion of B23/nucleophosmin or codepletion of SENP3 and SENP5, SUMO proteins accumulate within nucleoli. Importantly, depletion of SENP3 and SENP5 causes defects in ribosome biogenesis closely reminiscent of those observed in the absence of B23/nucleophosmin. Collectively, our findings indicated that control of SUMO deconjugation through SENP3 and SENP5 may be a major facet of B23/nucleophosmin function, and that disruption of SUMOylation may significantly contribute to the phenotypes observed after B23/nucleophosmin loss. Notably, SENP3 and SENP5 have slightly different roles in ribosome biogenesis, with SENP5 acting at the level of rRNA transcription and SENP3 acting at later stages of rRNA processing and ribosome assembly. To understand the role of SENP3 at a biochemical level, we have characterized its protein interaction partners within Xenopus egg extracts (XEEs). We found that SENP3 binds stably to three proteins that show sequence similarity to yeast ribosome assembly factors Rix1p, Ipi1p and Ipi3p. Mammalian homologues of these SENP3-interacting proteins are essential for ribosome biogenesis in tissue culture cells, further suggesting that they are previously unrecognized vertebrate homologues of the yeast Rix1 complex. Ran is a small GTPase that binds to a family of nuclear transport receptors in its GTP-bound state. Ran-GTP binding regulates the cargo binding and release of these receptors, so that the differential levels of Ran-GTP in the cytoplasm and nucleus determine the directionality of transport. SENP3 and the Rix1 complex bound 60S ribosomal particles through B23/Nucleophosmin under high Ran-GTP conditions. Under low Ran-GTP conditions, B23/nucleophosmin was lost and they bound instead to RanBP5, a nuclear import receptor. While there is no evidence that yeast Rix1 activity is integrated with Ulp1p function, our findings demonstrate that SENP3 and the vertebrate Rix1 complex are coupled, both physically and functionally. Our data also suggest that B23/nucleophosmin and the Ran pathway regulate SENP3 and the Rix1 complex in a novel, antagonistic fashion. Yeast Ulp2p is nucleoplasmic and not essential for vegetative growth, but it is important for chromosome segregation. Ulp2p acts particularly in disassembly of poly-SUMO chains. Human SENP6 similarly prefers substrates containing multiple SUMO-2/3 moieties. PolySUMOylated species are also substrates of SUMO-targeted ubiquitin ligases (STUbLs), leading to their proteasomal degradation. The RNF4 protein is a major STUbL in mammalian cells. We analyzed the mitotic role of SENP6 in mammalian cells. We found that SENP6-depleted HeLa cells have defects in metaphase chromosome congression, and they show characteristic changes in spindle morphology. We examined kinetochore composition to find the molecular basis of these phenotypes: we found that a subset of the inner kinetochore proteins became undetectable at the kinetochores of SENP6-depleted cells, including components of the CENP-H/I/K and CENP-O complexes. At the same time, changes in outer kinetochore composition closely mimicked phenotypes observed after loss of CENP-H/I/K components. Importantly, we found that the CENP-H and -I proteins were quantitatively degraded in the absence of SENP6 through a mechanism that requires both RNF4 and proteasome-mediated proteolysis. Together, these findings demonstrate a novel function of the SUMO pathway in inner kinetochore assembly, which finely balances the incorporation and degradation of components of the inner plate.